Field of the Invention
This invention pertains to systems and methods for amplifying and detecting nucleic acids. In one embodiment, it pertains to methods for monitoring a polymerase chain reaction (PCR) in a microfluidic system.
Discussion of the Related Art
The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. PCR is perhaps the most well-known of a number of different amplification techniques.
PCR is a powerful technique for amplifying short sections of DNA. With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three phase temperature cycle of denaturation of DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated so that there are enough copies to be detected and analyzed. In principle, each cycle of PCR could double the number of copies. In practice, the multiplication achieved after each cycle is always less than 2. Furthermore, as PCR cycling continues, the buildup of amplified DNA products eventually ceases as the concentrations of required reactants diminish. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide to Methods and Applications, M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990).
Real-time PCR refers to a growing set of techniques in which one measures the buildup of amplified DNA products as the reaction progresses, typically once per PCR cycle. Monitoring the accumulation of products over time allows one to determine the efficiency of the reaction, as well as to estimate the initial concentration of DNA template molecules. For general details concerning real-time PCR see Real-Time PCR: An Essential Guide, K. Edwards et al., eds., Horizon Bioscience, Norwich, U.K. (2004).
Several different real-time detection chemistries now exist to indicate the presence of amplified DNA. Most of these depend upon fluorescence indicators that change properties as a result of the PCR process. Among these detection chemistries are DNA binding dyes (such as SYBR® Green) that increase fluorescence efficiency upon binding to double stranded DNA. Other real-time detection chemistries utilize Foerster resonance energy transfer (FRET), a phenomenon by which the fluorescence efficiency of a dye is strongly dependent on its proximity to another light absorbing moiety or quencher. These dyes and quenchers are typically attached to a DNA sequence-specific probe or primer. Among the FRET-based detection chemistries are hydrolysis probes and conformation probes. Hydrolysis probes (such as the TaqMan® probe) use the polymerase enzyme to cleave a reporter dye molecule from a quencher dye molecule attached to an oligonucleotide probe. Conformation probes (such as molecular beacons) utilize a dye attached to an oligonucleotide, whose fluorescence emission changes upon the conformational change of the oligonucleotide hybridizing to the target DNA.
A number of commercial instruments exist that perform real-time PCR. Examples of available instruments include the Applied Biosystems PRISM 7500, the Bio-Rad iCylcer, and the Roche Diagnostics LightCycler 2.0. The sample containers for these instruments are closed tubes which typically require at least a 10 μl volume of sample solution. If the lowest concentrations of template DNA detectable by a particular assay were on the order of one molecule per microliter, the detection limit for available instruments would be on the order of tens of targets per sample tube.
More recently, a number of high throughput approaches to performing PCR and other amplification reactions have been developed, e.g., involving amplification reactions in microfluidic devices, as well as methods for detecting and analyzing amplified nucleic acids in or on the devices. Thermal cycling of the sample for amplification is usually accomplished in one of two methods. In the first method, the sample solution is loaded into the device and the temperature is cycled in time, much like a conventional PCR instrument. In the second method, the sample solution is pumped continuously through spatially varying temperature zones.
U.S. patent application Ser. No. 11/505,358, entitled, “Real-time PCR in micro-channels,” which is assigned to the assignee of this application and which is incorporated herein by this reference in its entirety, describes, among other things, a novel method to acquire real-time PCR data in a microfluidic system. One of the steps in that method is to capture an image of a fluorescent signal along the length of at least one microfluidic channel.
A conventional apparatus to capture an image of a fluorescent signal is illustrated in FIG. 1. As illustrated in FIG. 1, the light emitted from the material under study is collected by a high numerical aperture objective and the light is re-imaged onto a two-dimensional detector array.
A reason for using a high numerical aperture objective to collect luminescence is that the solid angle subtended is higher, and therefore the photon collection efficiency is higher, than that achieved using a low numerical aperture objective. In certain cases, collection efficiency may be an important parameter because, in certain cases, emitted light flux is often so low that signal levels at the detector are weak. Therefore, at least in certain cases, it is desirable to maximize collection efficiency.
The drawback of using a high numerical aperture microscope objective is that the imaged area is small. The effective field of view of a conventional fluorescence microscope imaging system might have a linear dimension of 1 mm or smaller. This becomes a problem when the region of interest on a microfluidic chip is larger (e.g., if the length and width are in the range of 10-100 mm).
One strategy to address the problem of imaging a large region of interest with high collection efficiency is to use an optical system with large diameter optics. This strategy has a benefit that most or all of the region of interest may be imaged simultaneously. An example of this approach is illustrated in U.S. Patent Application 2006/0006067, entitled, “Optical Lens System and Method for Microfluidic Devices,” which describes a multi-element lens system.
Another strategy would be to translate the sample holder with respect to the optical system or vice versa (e.g. in a raster pattern) to collect pixel data in series. An example of this approach is described in U.S. Pat. No. 5,631,734, entitled, “Method and Apparatus for Detection of Fluorescently Labeled Materials.” This patent describes a system for collecting fluorescence data from a substrate, for example a DNA microarray, in which the substrate is held by an x-y-z translation stage and translated in front of a microscope objective.
PCT publication WO 2005/075683 A1, entitled, “High Throughput Device for Performing Continuous-Flow Reactions,” describes a continuous-flow PCR device that uses a fused silica capillary wrapped into a helix around three temperature-controlled blocks. This publication shows a microscope objective lens being scanned transverse to the windings. Although the description is short on detail, presumably an entire optical imaging system, including lenses, beam-splitters, filters, and detectors, would have to be scanned along as well.
U.S. Pat. No. 5,928,907, entitled, “System for Real Time Detection of Nucleic Acid Amplification Products,” describes a system for real-time PCR monitoring that uses a fiber optic and a lens to capture fluorescence from a closed, Eppendorf-style sample tube. The sample tube volume was 200 ul, and the fiber optic and 8 mm diameter collection lens were fixed with respect to the tube, looking down through the top of the tube and the airspace over the sample solution.
U.S. Patent Application 2005/0069257 A1, “Fiber Lens with Multimode Pigtail” gives an example of a miniature lens system that is permanently affixed to the end of an optical fiber. Further examples of miniature fiber coupling systems can be found in product literature by Corning Inc. for lensed fibers, tapered fibers, and gradient index fibers and lenses. These devices are used typically in telecommunication equipment, for example, for coupling light from a semiconductor diode laser into an optical fiber, or for coupling light from one fiber into another fiber.